Genetic information of organisms in the earth are transferred by using nucleic acids comprising of four bases consisting of adenine (A), guanine (G), cytosine (C) and thymine (T) as a gene. Proteins are synthesized according to genetic informations of mRNA which is transcribed from DNA of gene. In that occasion, 64 types of codon consisting of 3 bases (43=64) correspond to 20 types of amino acids.
If novel nucleic acid base (X and Y, in which X and Y form specific base pair) can be created in addition to already known for bases (A, G, C, T), numbers of codon can be increased greatly (63=216). As a result, proteins containing non-natural amino acids can possibly be synthesized by matching the newly created codons with non-natural amino acids [J. D. Bain, et al. Nature, 356, 537-539 (1992)].
Heretofore, a pair of isocytosine and isoguanine has been reported as an artificial base pair except for A-T and G-C. Isoguanine tends to form base pair with thymine due to tautomerism of isoguanine [C. Switzer, et al. J. Am. Chem. Soc. 111, 8322-8323 (1989); C. Y. Switzer, et al. Biochemistry 32, 10489-10496 (1993)]. Several novel base pairs have been reported, but there were problems on recognition by polymerase and no practical use has known [J. A. Piccirilli, et al., Nature, 343, 33-37 (1990); J. Horlacher, et al. Proc. Natl. Acad. Sci. USA, 92, 6329-6333 (1995); J. C. Morales, et al., Nature struct. biol., 5, 954-959 (1998)].
Nucleic acid molecules having various functions were found by in vitro selection method [A. D. Ellington, et al. Nature 346, 818-822 (1990); C. Tuerk, et al. Science 249, 505-510 (1990)]. If the novel base pair X-Y hereinabove can be recognized by polymerases such as DNA polymerase, RNA polymerase and reverse transcriptase, the present in vitro selection method using 4 bases can be performed by using 6 bases, then possibility to create nucleic acid molecules having novel function, which could not be practically realized by using 4 bases, can be expected.
Further, creation of novel base pair has expected for treatment of hereditary diseases caused by gene abnormality, in which one or more base in the gene is replaced by different base.
We have studied extensively to created novel artificial nucleic acid base pairs, which could not form base pair with natural nucleic acid, but could selectively form base pair by themselves and could be recognized by various polymerases. We have found that formation of nucleic acid base pair could be inhibited by applying steric hindrance of nucleic acid base, and formation of selective base pair between newly designed nucleic acid bases could be made. Further, we have found that such the newly designed nucleic acids could be recognized by various natural polymerases.
For example, in order not to form base pair with thymine but to form steric hindrance with keto group at position-6 of thymine, 2-amino-6-(N,N-dimethylamino) purine (hereinafter designates as X), in which two bulky methyl groups are introduced in amino group at position-6 of 2,6-diaminopurine, is designed. As a result, the X does not form base pair with thymine, but bases such as pyridine-2-one (hereinafter designates as Y), an analog of thymine, in which oxo group at position-6 is replaced by hydrogen atom, can form base pair with X (refer to FIG. 1).
Further, we have synthesized DNA oligomer containing 2-amino-6-(N,N-dimethylamino)-9-(2′-deoxy-β-D-ribofuranosyl) purine (hereinafter designates as dX) and 3-(2′-deoxy-5′-triphosphoro-β-D-ribofuranosyl) pyridine-2-one (hereinafter designates as dYTP), and found that dYTP or its ribonucleotide (rYTP) could be incorporated selectively into DNA or RNA as a complementary strand of the above dX.
This compound could hinder base pairing with natural base such as thymine (or uridine) (refer to FIG. 2 b) and cytosine in some extent due to steric bulkiness of dimethylamino group in the base (dx in FIG. 2 a). However, this steric hindrance could affect to the neighboring bases, and simultaneously could give inferior effect on stacking between bases, and resulted low rate of incorporation of dYTP by Klenow fragment as well as insufficient suppression for incorporation of thymidine triphosphate (dTTP) to dx.
We have examined novel artificial base pair by considering not only steric hindrance but also electrostatic repulsion between bases and stacking action with the neighboring bases, and could obtain artificial base pair with superior selectivity.